Technical Abstract:
Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been realized to be the assay of choice among available techniques for mRNA quantification analysis. For conventional practice, housekeeping genes have been applied as internal reference for data normalization and analysis since the technology appeared. With increased concerns of variability of housekeeping genes in response to varied environmental conditions, there is no commonly accepted housekeeping gene reference available. Data processing referenced by housekeeping genes is basically depending upon discretion of independent researcher. Such obtained data from different experimental sets or sources are often not repeatable or reproducible. It is difficult to compare results from different experiments, conditions, or among different studies and different laboratories. Accurate data acquisition and data reproducibility remain challenging. Previously, we developed the first universal external RNA controls that can be applied for gene expression analysis using different platforms of microarray and qRT-PCR, including SYBR Green and TaqMan probe-based chemistry (Journal of Microbiological Methods 68:486-496). Based on this technology, we further developed a robust master equation derived from a set of universal RNA controls for simple and efficient absolute mRNA quantification analysis using qRT-PCR. Using Saccharomyces cerevisiae as an example, this presentation demonstrates independent performance of this control standard system under a toxic compound treatment condition. This newly developed quality control system using the master equation is robust, which simplifies conventional qRT-PCR procedures and increases data reliability, reproducibility, and throughput of the qRT-PCR assay.